The sarcoplasmic reticulum (SR) is a sub-cellular organelle responsible for regulating the Ca2+ concentration in the cytosol of muscle fibers. By hydrolysis of ATP, the SR network lowers the free Ca2+ concentration in the space surrounding the myofibrils to sub-micromolar levels, pumping Ca2+ into the lumen of the SR. The reduction of myoplasmic free Ca2+ concentration leads to muscle relaxation.
Muscle contraction is initiated by an action potential at the cell's surface membrane. This depolarization propagates down the transverse (T) tubules, which in turn triggers the release of Ca2+ stored in the SR and contraction. More particularly, calcium release channels (CRCs) in the SR called ryanodine receptors (RyRs) open and release Ca2+ from the SR into the intracellular cytoplasm of the cell. Release of Ca2+ into the cytoplasm from the SR increases cytoplasmic Ca2+ concentration. Open probability (Po) of the RyR receptor refers to the likelihood that the RyR channel is open at any given moment, and therefore capable of releasing Ca2+ into the cytoplasm from the SR.
There are three types of ryanodine receptors, all of which are highly-related Ca2+ channels: RyR1, RyR2, and RyR3. RyR1 is found predominantly in skeletal muscle as well as other tissues, while RyR2 is found predominantly in the heart as well as other tissues, and RyR3 is found in the brain as well as other tissues. The RyR channels are formed by four RyR polypeptides in association with four FK506 binding proteins (FKBPs), specifically FKBP12 (calstabin1) and FKBP12.6 (calstabin2). Calstabin1 binds to RyR1, calstabin2 binds to RyR2, and calstabin1 binds to RyR3. The FKBP proteins (calstabin1 and calstabin2) bind to the RyR channel (one molecule per RyR subunit), stabilize RyR-channel functioning, and facilitate coupled gating between neighboring RyR channels, thereby preventing abnormal activation of the channel during the channel's closed state.
Important advances have been made toward understanding the 3-dimensional structure of the ryanodine receptor (RyR)/Ca2+ release protein, and the possible functional role of other junctional SR proteins in excitation contraction coupling (ECC) in skeletal muscle. ECC differs in skeletal and cardiac muscle. In skeletal muscle, there appears to be a mechanical coupling between the dihydropyridine receptor (DHPR) found in the T-tubule membrane and the CRC or RyR found at the terminal end of the SR. In cardiac muscle, Ca2+ enters the cell during the action potential through the DHPR, and initiates Ca2+ release from the SR via a mechanism known as Ca2+-induced Ca2+ release. See e.g., Meissner, “Ryanodine receptor/Ca2+ release channels and their regulation by endogenous effectors,” Annu. Rev. Physiol. (1994), 56: 485-508; Dulhunty et al., “Ion channels in the sarcoplasmic reticulum of striated muscle,” Acta. Physiol. Scand. (1996), 156: 375-85; Halling et al., “Regulation of voltage-gated Ca2+ channels by calmodulin,” Sci. STKE. (2005); 2005: re15; Coronado et al., “Structure and function of ryanodine receptors,” Am. J. Physiol. (1994), 266: C1485-C1504; and Dulhunty et al., “Excitation-contraction coupling from the 1950s into the new millennium,” Clin. Exp. Pharmacol. Physiol. (2006), 33: 763-72.
A number of associated proteins regulate the activity of the SR ryanodine receptors. The DHPR and RyR appear to form a hub for a large macromolecular complex, which includes triadin and calsequestrin (on the luminal face of the SR), FKBP12 (skeletal muscle) and FKBP12.6 (cardiac muscle), calmodulin, Ca2+—CaM kinase (skeletal muscle), and protein kinase A (PKA) (cardiac muscle). Defective RyR-FKBP12.6 association has been implicated in heart failure, cardiomyopathy, cardiac hypertrophy, and exercise induced sudden cardiac death. It has been proposed that PKA phosphorylation of the cardiac RyR2 results in dissociation of FKBP12.6 from the Ca2+ release channel, which results in an increased channel open probability (Po), increased sensitivity to activation by Ca2+, and destabilization of the CRC. Alternatively, it has been proposed that abnormal Ca2+ handling by calsequestrin may lead to an increased Ca2+ leak and cardiac arrhythmias. The cardio-protective agent K201 (also known as JTV519) and the antioxidant edaravone appear to correct the defective FKBP12.6 control of RyR2 and improve function. However, the mechanism of action of K201 is controversial. One report has shown that K201 suppresses spontaneous Ca2+ release in ventricular myocytes independent of the presence of the FKBP12.6 protein, suggesting that the mode by which K201 decreases the Ca2+ leak from cardiac SR does not involve the FKBP12.6 protein. See Hunt et al., “K201 (JTV519) suppresses spontaneous Ca2+ release and [3H]ryanodine binding to RyR2 irrespective of FKBP12.6 association,” Biochem. J. (2007), 404: 431-38.
In addition, CRCs from both cardiac and skeletal muscle SR are rich in thiol groups, and therefore, are strongly regulated by thiol reagents. It has been shown that oxidation of these thiol groups results in increased Ca2+ release rates from SR vesicles, increased open probability of the reconstituted CRC, and increased high infinity ryanodine binding to the SR, while reduction of the disulfide(s) formed results in decreased activity. There are also a large number of non-thiol reagents known to either activate or inhibit RyR1 and/or RyR2. Among those compounds that activate the RyR/CRC are methylxanthines such as caffeine, plant alkaloids such as ryanodine, polyamines such as polylysine, quinone such as doxorubicin, and phenols such as 4-chloro-m-cresol (4-CmC). Among the non-thiol RyR/CRC inhibitors are local anesthetics such as tetracaine and procaine, the poly-unsaturated fatty acids such as docosahexaenoic acid (DHA). However, these reagents are physiologically and pharmacologically diverse, and their exact mode of action is not clear. Accordingly, the art desires better understanding of calcium release mechanisms for developing a broader class of RyR/CRC activators and inhibitors.